The present invention relates to peptide analogues which inhibit prenylated pyrophosphate-consuming enzymes. Prenylated pyrophosphate consuming enzymes are understood to comprise protein:farnesyl transferases, protein:geranylgeranyl transferases and several other enzymes involved in the biosynthesis of terpenes, such as farnesyl pyrophosphate synthase, squalene synthase and geranylgeranyl pyrophosphate synthase.
Prenylated pyrophosphates, i.e. all-trans-farnesyl pyrophosphate (FPP) and all-trans-geranylgeranyl pyrophosphate (GGPP), are substrates for a number of different enzymes. Protein:farnesyl transferase (PFT) is an enzyme which uses FPP to catalyse the farnesylation of cysteine residues near the C-terminus of certain proteins. The enzyme geranylgeranyl pyrophosphate synthase (GGPPS) uses FPP as a substrate in the production of geranylgeranyl pyrophosphate (GGPP), which is subsequently used in the synthesis of geranylgeranylated proteins mediated by the enzymes protein:geranylgeranyl transferase 1 and 2 (PGGT-1 and 2).
Several of these prenylated (i.e. farnesylated and/or geranylgeranylated) proteins were identified as belonging to groups of related proteins: e.g. the nuclear lamins, low molecular weight GTP binding proteins (G-proteins), such as the ras-oncogene proteins and the xcex3-subunit of heterotrimeric G-proteins (Schafer et al., Science 245 (1989) 379-385). Lamins and p21ras proteins, 188 or 189 amino acid proteins which possess the consensus CaaX motif (C=cysteine, a=any amino acid having an aliphatic side chain and X=methionine, serine, glutamine or alanine) at the C-terminus, are farnesylated. Other small G-proteins, that have the consensus CaaL motif (L=leucine) such as Rho and several members of the rab proteins having C-terminal CC/CXC motifs, and also heterotrimeric G-protein xcex3-subunits are geranylgeranylated.
G-proteins are involved in the receptor-mediated transduction of signals (such as growth modulation signals) over the plasma membrane, and other prenylated proteins, not yet identified, may have a function in cell cycle progression. The prenylation of these proteins seems to play a role in their association with membranes and nuclear envelopes, where they are processed further and/or perform their function. This was shown for example by blocking the mevalonate synthesis by HMG-CoA reductase inhibitors, which prevented proteolytic processing of the lamin A precursor (Beck et al., J. Cell. Biol. 110 (1990) 1489-1499) or resulted, in other studies, in the accumulation of non-prenylated p21ras precursor and the loss of transforming activity of oncogenic ras proteins. A review of the post-translational modification of proteins by isoprenoids in mammalian cells is given by Maltese W. A. in FASEB J. 4 3319-3329 (1990). The latter observation triggered the search for specific inhibitors of the farnesylation of p21ras in order to prevent its action in cells, where over-expression of this protein leads to tumour development, such as in colon carcinomas.
We have shown that FPP-analogues, which are in vitro inhibitors of PFT and/or PGGT-1 are able to inhibit the proliferation of human arterial smooth muscle cells in culture (Cohen et al. Biochem. Pharm. 57 (1999), 365-373) and are therefore potential inhibitors of the process of restenosis after percutaneous transluminal coronary angioplasty. The proliferation of smooth muscle cells plays a major role in the latter process.
Very recently it was shown that inhibition of the cellular GGPP synthesis in specific bone cells (osteoclasts) is the possible mechanism of action of anti-osteoporosis drugs (Van Beek, E. Biochem. Biophys. Res. Commun. 255, (1999), 491-494 and Van Beek, E. J. Bone and Min. Res. 14 (1999) , 722-729). So, inhibitors of GGPPS and/or PGGT-1 and/or -2 may be active as anti-osteoporosis drugs as well.
Furthermore, G-proteins of the rab-family are involved in the regulation of intracellular protein traffic and secretion. In addition, prenylated proteins seem to play a role in the translational control of HMG-CoA reductase, the rate limiting enzyme of the isoprene and subsequent cholesterol biosynthesis. Squalene synthase (SS), the first pathway-specific enzyme in the biosynthesis of cholesterol, catalyses the reductive dimerisation of two molecules of FPP to produce squalene. Much attention has been directed to the development of inhibitors of SS with the aim to lower elevated blood plasma levels of cholesterol, one of the risk factors for cardiovascular diseases.
EP-A-540782 describes inhibitors for protein:farnesyl transferase based on prenyl pyrophosphate analogues. A review of new therapeutic methods based on protein:farnesyl transferase and inhibitors thereof are described by Leonard in J. Med. Chem. 40 (1997), 2971-2990. Furthermore, we recently (Overkleeft et al. Tetrahedron Let. 40 (1999) 4103-4107) described inhibitors of protein:prenyl transferases based on binding in the CaaX pocket of the enzyme concerned (see also EP-A-1028117).
Novel analogues of prenyl pyrophosphate have been found according to the invention, which are inhibitors of prenylated pyrophosphate consuming enzymes (e.g. PFT, PGGT-1 and 2, GGPPS and SS) presumably by binding in the FPP pocket. The analogues are defined in the appending claims with reference to formulae 1 (Axe2x80x94Bxe2x80x94D) and 2 (Axe2x80x94Bxe2x80x94Bxe2x80x2xe2x80x94D). The novel prenylated pyrophosphate-based inhibitors contain unnatural amino acid moieties which have the potential to be unsusceptible towards enzymatic degradation. These novel inhibitors are composed of amino acid derivatives which are ideally suited for the construction of a compound library via a combinatorial approach. Furthermore their peptide-like structure allows for easy access to bisubstrate analogues, in which the new FPP analogues are connected to CaaX box analogues, as defined in the appending claims.